WO2021013257A1 - Procédé de détection multi-cible crispr et kit de test associé - Google Patents

Procédé de détection multi-cible crispr et kit de test associé Download PDF

Info

Publication number
WO2021013257A1
WO2021013257A1 PCT/CN2020/104588 CN2020104588W WO2021013257A1 WO 2021013257 A1 WO2021013257 A1 WO 2021013257A1 CN 2020104588 W CN2020104588 W CN 2020104588W WO 2021013257 A1 WO2021013257 A1 WO 2021013257A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
detection system
container
guide rna
cas protein
Prior art date
Application number
PCT/CN2020/104588
Other languages
English (en)
Chinese (zh)
Inventor
王金
李诗渊
Original Assignee
上海吐露港生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海吐露港生物科技有限公司 filed Critical 上海吐露港生物科技有限公司
Priority to US17/629,545 priority Critical patent/US20220267847A1/en
Priority to CN202080052987.5A priority patent/CN114174535A/zh
Priority to EP20843278.1A priority patent/EP4006170A4/fr
Publication of WO2021013257A1 publication Critical patent/WO2021013257A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to a CRISPR multi-target detection method and its kit.
  • Cas12 and Cas13 proteins can be used for nucleic acid detection based on their bypass (or trans) cleavage activity, that is, under the guidance of an artificially designed guide RNA, they bind to nucleic acid fragments of a specific sequence, and then cut single-stranded nucleic acid probes This produces a detectable signal.
  • the difference between Cas12 and Cas13 is that Cas13 protein binds to RNA targets and cleaves RNA probes, while Cas12 binds to DNA targets and cleaves single-stranded DNA probes.
  • RNA reporter probe used in the Cas13 detection process has a greater risk of being degraded by RNase, which results in a higher background signal value of the detection system.
  • the Cas12-based detection method has advantages because it does not require in vitro transcription or RNA reporter probes.
  • the methods and techniques for simultaneously detecting multiple existing targets in one detection system are very important for the expanded application of in vitro diagnostics.
  • a multi-target detection method has been developed based on the Cas13 protein. The key is to take advantage of the different bypass cleavage activities of Cas13 proteins from different species for different RNA reporter probes.
  • Cas12 there is no research report on this characteristic of Cas12; therefore, it is necessary to develop a new method that uses Cas protein for multi-target nucleic acid detection.
  • the purpose of the present invention is to provide a multi-target detection method and kit based on CRISPR-Cas protein.
  • a detection system for detecting target nucleic acid molecules comprising:
  • the guide RNA-reporter nucleic acid composite probes have the structure shown in formula Ia, Ib, Ic or Id,
  • Z1 is the first stem-loop structure region
  • Z2 is no or nucleic acid linking region
  • Z3 is the guide RNA region
  • Z5 is a single-stranded nucleic acid to be cleaved with a detectable label, wherein the detectable label presents different detection states when the single-stranded nucleic acid to be cleaved is cut and uncut, and thus is detected;
  • Z3 and Z5 form a complementary paired double-stranded structural region; and when the target nucleic acid exists in the detection system, Z3 and Z5 do not form the complementary paired structure. Double-stranded structure region;
  • Z4 is none, or a chemical bond or connecting zone used to connect Z3 and Z5;
  • n is a positive integer n ⁇ 1;
  • Cas protein which is a Cas protein with bypass single-stranded nucleic acid cleavage activity.
  • the Cas protein is selected from the following group: Cas12 type, Cas13a type, Cas13b type, Cas14 type, Cas ⁇ , or a combination thereof.
  • the guide RNA-reporter nucleic acid composite probe has a structure as shown in Formula Ia or Ib.
  • the guide RNA-reporter nucleic acid composite probe has a structure shown in Formula Ic or Formula Id.
  • the Cas12 type is selected from the following group: Cas12a, Cas12b, Cas12d, Cas12g, Cas12i, or a combination thereof.
  • the guide RNA-reporter nucleic acid composite probe has a structure of formula IIa:
  • Z1, Z2, Z3, Z4 and Z5 are as described above,
  • the "Z1-Z2-Z3" in formula Ia, formula IIa, formula Ic, formula Ib and "Z3-Z2-Z1" in formula Id are from 5'to 3' direction.
  • the guide RNA-reporter nucleic acid composite probe has a structure of formula IIc:
  • Z1, Z2, Z3, Z4 and Z5 are as described above,
  • the complementary paired double-stranded structural region includes a double-stranded structural region formed by partial or complete complementary pairing of Z3 and Z5.
  • the complementary paired double-stranded structural region is a double-stranded structural region formed by all complementary pairings of Z3 and Z5.
  • the Z1, Z2, and Z3 are used to guide the Cas protein to bind to the target nucleic acid.
  • the Z1 is used to bind or anchor Cas protein.
  • the guide RNA region guides the Cas protein to bind to the target nucleic acid through complementary pairing with the target nucleic acid.
  • Z3, Z4, and Z5 form a second stem-loop structure region, where Z4 is a loop region (including simple structure and complex structure loop region).
  • said Z1 is substantially or entirely composed of RNA.
  • the Z3 is substantially or entirely composed of RNA.
  • the guide RNA-reporter nucleic acid composite probe is single-stranded.
  • the stem-loop structure in Z1 is a crRNA (or CRISPR RNA) stem-loop structure.
  • the length of Z1 is 10-300 nt, preferably 19-100 nt, and more preferably 19-91 nt.
  • the Z2 is a nucleic acid linking region without or with a length of 0-20 nt.
  • the detectable label is a fluorescent group
  • Z5 has a fluorescent group
  • the Z4 and/or Z5 also has a quenching group, if and only if the single strand is to be cleaved
  • the fluorescent signal emitted by the fluorescent group can be detected;
  • the fluorescent groups are different from each other so that they can be distinguished.
  • the detectable label is a fluorescent group
  • Z5 has a quenching group
  • the Z4 and/or Z5 also has a fluorescent group, if and only if the When the single-stranded nucleic acid to be cut is cleaved, the fluorescent signal emitted by the fluorescent group can be detected.
  • the detectable label is a fluorescent group, wherein the fluorescent group is located in any zone of Z5, Z4 and Z3, and the quenching group is located in any zone of Z5, Z4 and Z3 Section, when and only when the single-stranded nucleic acid to be cleaved is cleaved, the fluorescent signal emitted by the fluorescent group can be detected.
  • the fluorescent group and the quenching group are not located at Z3 at the same time.
  • the Z3 contains a nucleic acid sequence that can guide the Cas protein to specifically bind to a target nucleic acid molecule.
  • the length of Z3 is 15-50nt, preferably 16-40nt, more preferably 16-34nt.
  • the Z1, Z2 and Z3 are all RNA nucleic acid sequences.
  • the Z4 is a DNA and/or RNA nucleic acid sequence.
  • the Z5 is a DNA single-stranded nucleic acid sequence, or an RNA single-stranded nucleic acid sequence, or a nucleic acid sequence of both RNA and DNA.
  • the Z5 contains nucleotides based on natural bases, or nucleotides based on natural bases and non-natural bases.
  • the nucleotides include ribonucleic acid, deoxyribonucleic acid, peptide nucleic acid, or a combination thereof.
  • the natural base is selected from the following group: A, T, C, G, U, and I.
  • the length of the Z5 is 3-50 nt, preferably 4-30 nt, more preferably 6-12 nt.
  • the labels carried in the Z5 are a fluorescent group and a quenching group.
  • the fluorescent group and the quenching group are independently located at the 5'end, 3'end and/or middle of the Z5.
  • each guide RNA-reporter nucleic acid composite probe has different fluorescent groups and different or the same quenching groups between each pair.
  • the detection system also contains m target nucleic acid molecules to be detected, where m is a positive integer and m ⁇ n.
  • the target nucleic acid molecule includes a target nucleic acid molecule derived from the group consisting of plants, animals, insects, microorganisms, viruses, or combinations thereof.
  • the target nucleic acid is artificially synthesized or naturally occurring nucleic acid.
  • the target nucleic acid includes wild-type or mutant nucleic acid.
  • the target nucleic acid molecule is target DNA or RNA.
  • the target DNA includes DNA without reverse transcription or DNA obtained by reverse transcription or amplification of RNA (for example, cDNA, etc.).
  • the target RNA includes RNA that has not been transcribed or is transcribed from DNA.
  • the detection includes: qualitative detection or quantitative detection.
  • the detection system further contains (c) buffer.
  • the detection system further contains the target nucleic acid molecule to be detected.
  • the detection system further contains reagents for nucleic acid amplification reactions.
  • the detection system further contains:
  • the concentration of the target nucleic acid molecule to be detected in the system to be detected is 1 ⁇ 10 -9 nM to 1 ⁇ 10 3 nM; preferably 1 ⁇ 10 -8 nM to 1 ⁇ 10 2 nM.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1 to 1 ⁇ 10 15 copies/ml, preferably 1 to 10 10 copies/ml, more preferably 1 To 10 5 copies/ml.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1 to 1000 copies/ml, preferably 1 to 100 copies/ml, more preferably 1 to 10 copies/ml. Ml.
  • the molar ratio of each guide RNA-reporter nucleic acid composite probe to the corresponding target nucleic acid molecule is 1:1 to 10 14 :1, preferably 10:1 to 10 5 :1, more preferably 20:1 to 10 3 :1.
  • the Cas protein is selected from the group consisting of Cas12a, Cas12b, Cas12d, Cas12g, Cas12i, Cas13a, Cas13b, Cas14 and Cas ⁇ .
  • the Cas12a protein is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a and Lb4Cas12a.
  • the Cas12a protein is LbCas12a or FnCas12a.
  • the Cas12b protein is selected from the group consisting of AaCas12b, AacCas12b, AapCas12b, AbCas12b, AkCas12b, AmCas12b, BhCas12b, BsCas12b, EbCas12b and LsCas12b.
  • the Cas12g protein is Cas12g1.
  • the Cas12i protein is Cas12i1 or Cas12i2.
  • the Cas13a protein is selected from the group consisting of LshCas13a, LwaCas13a, LbaCas13a, LseCas13a, LbmCas13a, LbnCas13a, CamCas13a, CgaCas13a, Cga2Cas13a, PprCas13a, LweCas13a, Lwa2Cas13a, Rbf13Casa13, CasabuCas13a, RbfCas13a, RbfCas13a, LbnCas13a, Cga2Cas13a, PprCas13a, LweCas13a, Lwa2Cas13a, Rbf13Casa13, CasbuCas13a, Rbf13a, LbnCas13a.
  • the Cas13b protein is selected from the group consisting of BzoCas13b, PinCas13b, PbuCas13b, AspCas13b, PsmCas13b, RanCas13b, PauCas13b, PsaCas13b, Pin2Cas13b, CcaCas13b, PguCas13b, PspCas13b, PigCas13b and Pin.
  • the Cas14 protein is selected from the group consisting of Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h and Cas14u.
  • the Cas ⁇ protein is selected from the group consisting of Cas ⁇ -1, Cas ⁇ -2 and Cas ⁇ -3.
  • n is a positive integer between 2 and 200; preferably, n is a positive integer between 2 and 100; more preferably, n is a positive integer between 2 and 20; more preferably Ground, n is a positive integer between 2-10.
  • kits for detecting a target nucleic acid molecule comprising:
  • Z1 is the first stem-loop structure region
  • Z2 is no or nucleic acid linking region
  • Z3 is the guide RNA region
  • Z5 is a single-stranded nucleic acid to be cleaved with a detectable label, wherein the detectable label presents different detection states when the single-stranded nucleic acid to be cleaved is cut and uncut, and thus is detected;
  • Z3 and Z5 form a complementary paired double-stranded structural region; and when the target nucleic acid exists in the detection system, Z3 and Z5 do not form the complementary paired structure. Double-stranded structure region;
  • Z4 is none, or a chemical bond or connecting zone used to connect Z3 and Z5;
  • n is a positive integer n ⁇ 1;
  • the second container and the Cas protein in the second container, the Cas protein is a Cas protein with bypass single-stranded nucleic acid cleavage activity
  • the target nucleic acid molecule is target DNA and/or target RNA.
  • first container, the second container and the third container may be the same container or different containers.
  • the kit further includes:
  • the detection is used to simultaneously detect two or more different target nucleic acid molecules.
  • the detection system further contains reagents for nucleic acid amplification reactions.
  • the fourth container, the fifth container and the sixth container may be the same container or different containers.
  • two, more or all of the first container to the sixth container may be the same container or different containers.
  • a method for detecting target nucleic acid molecules in a sample which includes the following steps:
  • the guide RNA-reporter nucleic acid composite probe is cleaved by the Cas protein, which means that there is a corresponding target nucleic acid molecule in the sample; and the guide RNA-reporter nucleic acid composite probe is not cleaved by the Cas protein, it means that There is no corresponding target nucleic acid molecule in the sample.
  • the sample to be tested includes a sample that has not been amplified and a sample that has undergone amplification (or nucleic acid amplification).
  • the sample to be tested is a sample obtained through amplification.
  • the nucleic acid amplification method is selected from the following group: PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification Increase, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.
  • the PCR includes high temperature PCR, normal temperature PCR, or low temperature PCR.
  • the detection in step (ii) includes fluorescence detection.
  • the fluorescence detection method uses a microplate reader or a fluorescence spectrophotometer or a fluorescence quantitative PCR instrument for detection.
  • the method is an in vitro detection method.
  • the sample is an in vitro or ex vivo sample.
  • the method is non-diagnostic and non-therapeutic.
  • the method is diagnostic.
  • Figure 1 shows a schematic diagram of the guide RNA-reporter nucleic acid composite probe (Formula Ia).
  • F represents a fluorescent group (or other detectable label)
  • Q is a quenching group (or other quenching functional group for quenching the F signal).
  • FIG. 2 shows the results of CRISPR multi-target detection.
  • Figure 2A shows the colors of 8 different samples
  • Figure 2B is the Green channel: FAM fluorescence is detected, and the detected target sequence is DNMT1-3 sites
  • Figure 2C is the Orange channel: ROX fluorescence is detected, and the detected target sequence is detected. Is the sry site).
  • FIG 3 shows a schematic diagram of another guide RNA-reporter nucleic acid composite probe (formula Ib).
  • F represents a fluorescent group (or other detectable label)
  • Q is a quenching group (or other quenching functional group for quenching the F signal).
  • FIG 4 shows a schematic diagram of another guide RNA-reporter nucleic acid composite probe (formula Ic).
  • F represents a fluorescent group (or other detectable label)
  • Q is a quenching group (or other quenching functional group for quenching the F signal).
  • FIG. 5 shows a schematic diagram of another guide RNA-reporter nucleic acid composite probe (formula Id).
  • F represents a fluorescent group (or other detectable label)
  • Q is a quenching group (or other quenching functional group for quenching the F signal).
  • Figure 6 shows the structure of the guide RNA-reporter nucleic acid composite probe in Example 2 or Example 3.
  • Figure 7 shows the results of the Cas12b multi-target detection test.
  • Figure 8 shows the results of the Cas14a1 multi-target detection test.
  • the inventors developed a method based on CRISPR technology that can simultaneously detect multiple target nucleic acid molecules in the same detection system for the first time.
  • the present inventors developed a guide RNA-reporter nucleic acid composite probe in which the first stem-loop structure region of RNA, the guide RNA region, the connecting region, and the fluorescent group and quenching group are connected in series.
  • CRISPR refers to clustered regularly spaced short palindromic repeats (clustered regularly interspaced short palindromic repeats), which are part of the immune system of many prokaryotes.
  • Cas protein refers to CRISPR-associated protein, which is a related protein in the CRISPR system.
  • Cas12a (formerly known as “Cpf1”) refers to a crRNA-dependent endonuclease, which is a V-A type enzyme in the CRISPR system classification.
  • Cas12b and “C2c1” are used interchangeably and refer to the sgRNA-dependent endonuclease, which is a V-B type enzyme in the CRISPR system classification.
  • Cas12c and “C2c3” are used interchangeably and refer to tracrRNA:crRNA (or sgRNA)-dependent endonuclease, which is a V-C type enzyme in the CRISPR system classification.
  • Cas12d and “CasY” are used interchangeably and refer to scoutRNA:crRNA-dependent endonuclease, which is a V-D type enzyme in the CRISPR system classification.
  • Cas12g refers to a tracrRNA:crRNA (or sgRNA) dependent RNase, which is a V-G type enzyme in the CRISPR system classification.
  • Cas12i refers to a crRNA-dependent endonuclease, which is a type V-I enzyme in the CRISPR system classification.
  • Cas13a and C2c2 are used interchangeably and refer to crRNA-dependent endonucleases, which are type VI-A enzymes in the CRISPR system classification.
  • Cas13b refers to a crRNA-dependent endonuclease, which is a VI-B type enzyme in the CRISPR system classification.
  • Cas14 refers to a tracrRNA:crRNA (or sgRNA) dependent endonuclease, which is a V-F type enzyme in the CRISPR system classification.
  • Cas ⁇ and Cas12j are used interchangeably and refer to crRNA-dependent endonucleases, which belong to the V-type enzyme in the CRISPR system classification.
  • PCR refers to "polymerase chain reaction", a method used to amplify large amounts of DNA fragments of interest.
  • guide RNA-reporter nucleic acid composite probe of the present invention As used herein, the terms "guide RNA-reporter nucleic acid composite probe of the present invention”, “composite probe of the present invention”, and “probe of the present invention” are used interchangeably and refer to the target nucleic acid described in the present invention.
  • Molecular probes include guide RNA-reporter nucleic acid composite probes with Ia or Ib. It should be understood that the term also includes different forms of pairing, partial pairing or no pairing formed between Z3 and Z5 in the composite probe. For example, formula IIa is the state where Z3 and Z5 in the composite probe of formula Ia of the present invention form a pair.
  • a guide RNA-reporter nucleic acid composite probe with a novel structure is provided.
  • a representative composite probe has the structure shown in formula Ia,
  • Z1, Z2, Z3, Z4, and Z5 are as described above.
  • Z1, Z2, Z3, Z4, and Z5 are as described above.
  • the composite probe of formula Ic or formula Id is used in conjunction with the Cas protein of Cas13b type.
  • the labels carried in the Z5 are a fluorescent group and a quenching group, and the fluorescent group and the quenching group are each independently located at the 5'end of the nucleic acid probe , 3'end and/or middle.
  • RNA-reporter nucleic acid composite probe the structure of a representative guide RNA-reporter nucleic acid composite probe is shown in Figure 1.
  • a DNA sequence is added to the 3'end of the guide RNA, a quenching group (Q) is added in the middle, and a fluorescent group (F) is added to the 3'end.
  • Q quenching group
  • F fluorescent group
  • the sequence of the composite probe not only functions as a guide RNA, but also functions as a fluorescent probe.
  • the terminal sequence (Z5) of the DNA is complementary to a part of the sequence base (Z3) of the guide RNA to form a hairpin structure.
  • the composite probe of the present invention binds to the Cas12a protein and binds to the target sequence.
  • the hairpin structure (that is, the pairing structure of Z3 and Z5 will untie) will open, and the side of Cas12a will be activated Pathway single-stranded DNA cleavage activity, thereby cleaving the DNA part of the composite probe (such as Z5), causing the fluorescent group to be separated from the quenching group, thereby causing the quenching group to lose its quenching function and emit fluorescence.
  • the Cas12 bypass cleavage activity activated by other targets will not affect the unbound target.
  • the composite probes are chopped and will not interfere with each other.
  • reaction system for detecting one or more (especially simultaneous detection of multiple) target nucleic acid molecules, the reaction system comprising:
  • n kinds of the guide RNA-reporter nucleic acid composite probes of the present invention preferably, n is 2-500 or 2-200;
  • Cas protein which is a Cas protein with bypass single-stranded nucleic acid cleavage activity.
  • the detection system provided by the present invention can detect m target nucleic acid molecules to be detected, where m is a positive integer and m ⁇ n.
  • the detection includes: qualitative detection or quantitative detection.
  • the detection system further contains:
  • the concentration of the target nucleic acid molecule to be detected in the system to be detected is 1 ⁇ 10 -9 nM to 1 ⁇ 10 3 nM; preferably 1 ⁇ 10 -8 nM to 1 ⁇ 10 2 nM.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1 to 1 ⁇ 10 15 copies/ml, preferably 1 to 10 10 copies/ml, more preferably 1 To 10 5 copies/ml.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1 to 1000 copies/ml, preferably 1 to 100 copies/ml, more preferably 1 to 10 copies/ml. Ml.
  • the molar ratio of each guide RNA-reporter nucleic acid composite probe to the corresponding target nucleic acid molecule is 1:1 to 10 14 :1, preferably 10:1 to 10 5 :1, more preferably 20:1 to 10 3 :1.
  • a kit for detecting one or more target nucleic acid molecules (especially detecting multiple simultaneously) is provided, and the kit includes:
  • n a positive integer of 2-500 or 2-200
  • the second container and the Cas protein in the second container, the Cas protein is a Cas protein with bypass single-stranded nucleic acid cleavage activity
  • the detection system further contains reagents for nucleic acid amplification reactions. That is, in the detection system of the present invention, the target nucleic acid molecule can be amplified, and the amplified target nucleic acid molecule can be detected, which has the function of signal amplification.
  • the fourth container, the fifth container, and the sixth container may be the same container or different containers.
  • two, more or all of the first container to the sixth container may be the same container or different containers.
  • a method for simultaneously detecting multiple target nucleic acid molecules in a sample which includes the following steps:
  • the guide RNA-reporter nucleic acid composite probe is cleaved by the Cas protein, which means that there is a corresponding target nucleic acid molecule in the sample; and the guide RNA-reporter nucleic acid composite probe is not cleaved by the Cas protein, it means that There is no corresponding target nucleic acid molecule in the sample.
  • the sample to be tested includes unamplified samples and amplified (or nucleic acid amplified) samples, and may also include untranscribed samples and transcribed samples.
  • the nucleic acid amplification method is selected from the group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription mediator Guided amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.
  • the PCR includes high temperature PCR, normal temperature PCR, and/or low temperature PCR.
  • the detection in step (ii) includes fluorescence detection.
  • the fluorescence detection method uses a microplate reader or a fluorescence spectrophotometer for detection.
  • the method is an in vitro detection method.
  • the sample is an in vitro or ex vivo sample.
  • the method is non-diagnostic and non-therapeutic.
  • the multi-target detection method of the present invention realizes the detection of multiple target nucleic acid molecules at the same time with extremely high sensitivity in the same detection system, and can detect nucleic acid molecules (such as DNA) with a concentration of 10 -17 M.
  • Multi-purpose It can detect different nucleic acid samples, including DNA samples and RNA samples.
  • the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 1.
  • two kinds of probes are designed and synthesized, the sequence is as follows:
  • the target sequence needs to be amplified.
  • PCR or any other amplification method can be used.
  • isothermal LAMP amplification is used.
  • LAMP amplification reaction After heating male saliva at 95°C for 10 minutes, it serves as a template. The total volume of each reaction system is 20 ⁇ L, and two types of primers are added to respectively amplify the gene DNMT1-3 on the autosomes and the gene sry unique to the male Y chromosome (see primer table 2 for sequence). The specific primer amounts are 1.6 ⁇ M FIP and BIP, 0.2 ⁇ M F3 and B3, 0.4 ⁇ M LoopF and LoopB.
  • the kit used for the LAMP reaction is LAMP Kit (NEB).
  • the LAMP reaction program is 65°C for 40 minutes. The above products are called DNM and sry.
  • IS6110-1 fragment was amplified using the Mycobacterium tuberculosis genome as a template, and the amplified product was called IS-1.
  • Probes add 0.5 ⁇ L each (3 probes of 10 ⁇ M)
  • Figure 2B is the Green channel, which detects FAM fluorescence against the DNM target.
  • the samples that can detect significant rising curves are samples 2, 5, 6 and 8, respectively;
  • Figure 2C is the Orange channel, which is The ROX fluorescence of the sry target was detected, and samples 4, 6, 7 and 8 could be detected with a significant rising curve.
  • the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 6, which consists of tracrRNA and crRNA reporter nucleic acid probes.
  • Figure 6 the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 6, which consists of tracrRNA and crRNA reporter nucleic acid probes.
  • two kinds of probes are designed and synthesized, the sequence is as follows:
  • the transcription template was prepared by annealing T7-crRNA-F with the synthetic oligonucleotide Cas12b_tracrRNA (Table 3). Specifically, the paired oligonucleotides (4 ⁇ M) were annealed in 1 ⁇ PCR buffer (Transgen Biotech) with a total volume of 50 ⁇ L, and then an annealing procedure was performed: initial denaturation at 95°C for 5 minutes, and then cooling from 95°C to At 20°C, use a thermal cycler to reduce 1°C per minute. T7 high-yield transcription kit was used to synthesize tracrRNA, and the reaction was carried out at 37°C overnight (about 16h). Use DNase I to process template DNA, then use RNA purification and concentration kit to purify RNA, and quantify it with NanoDrop 2000C, and store it in a refrigerator at -80°C.
  • Cas12b_tracrRNA SEQ ID NO: 23
  • the target sequence needs to be amplified.
  • PCR or any other amplification method can be used.
  • the PCR amplification method is used.
  • PCR reaction separately extract Salmonella genomic DNA and E. coli O157 genomic DNA as templates for PCR amplification.
  • the total volume of each reaction system is 20 ⁇ L, and two types of primers are added to respectively amplify the specific fragments in Salmonella (product named SE) and E. coli O157 (product named O157) (see primer table 4 for sequence) .
  • the PCR reaction program is 95°C for 2 minutes, and then 35 cycles of 98°C for 10s, 60°C for 15s, and 72°C for 10s. After the PCR is completed, the product is directly used in the Cas12b reaction.
  • reaction samples There are two reaction samples, the difference is that the added template is different, they are the PCR amplification products of SE and O157 respectively, and the reaction system with sterile water is used as a negative control.
  • the fluorescence quantitative PCR instrument used in this reaction is ABI StepOne Plus, and the detected signal is FAM fluorescence.
  • the result is shown in Figure 7.
  • the FAM fluorescence signal of the reaction group added with the SE template is very low, while the FAM fluorescence signal of the reaction group added with the O157 template increases rapidly , And the signal intensity of the SE group formed a significant difference.
  • results of ROX fluorescence detection using a microplate reader show that the O157 template cannot activate the cleavage of the SE reporter nucleic acid probe, while the SE template can activate the cleavage of the SE reporter nucleic acid probe.
  • the above results show that once the corresponding target exists in the detection system, the corresponding fluorescent signal can be detected, which is consistent with the expected result; on the contrary, the existence of a certain target can be judged according to the type of the detected fluorescent signal.
  • the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 6, which consists of tracrRNA and crRNA reporter nucleic acid probes.
  • Figure 6 the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 6, which consists of tracrRNA and crRNA reporter nucleic acid probes.
  • two kinds of probes are designed and synthesized, the sequence is as follows:
  • tracrRNA with Cas14a1 was synthesized and cloned into the pUC57 vector. Then use T7-crRNA-F and Cas14a-tracr-R primer pair (Table 6) for amplification, and the amplified product is purified and used to transcribe tracrRNA.
  • the tracrRNA was synthesized using 200ng transcription template and T7 high-yield transcription kit, and the reaction was carried out overnight (about 16h) at 37°C.
  • Use DNase I to process template DNA, then use RNA purification and concentration kit to purify RNA, and quantify it with NanoDrop 2000C, and store it in a refrigerator at -80°C.
  • Synthetic Cas14a1 tracrRNA sequence with T7 promoter (SEQ ID NO: 32):
  • Tris buffer solution 50mM Tris-HCl[pH 8.3], 75mM KCl, 3mM MgCl 2
  • Tris buffer solution 50mM Tris-HCl[pH 8.3], 75mM KCl, 3mM MgCl 2
  • an annealing reaction is performed on a PCR machine. Initial denaturation at 85°C for 5 minutes, then cooling from 85°C to 25°C, using a thermal cycler to reduce 3°C every minute.
  • the complex probe after annealing can be used in the cleavage reaction of Cas14 to detect the target nucleic acid.
  • the target sequence needs to be amplified.
  • PCR or any other amplification method can be used.
  • the PCR amplification method is used.
  • PCR reaction separately extract Salmonella genomic DNA and E. coli O157 genomic DNA as templates for PCR amplification.
  • the total volume of each reaction system is 20 ⁇ L, and two types of primers are added for amplification. In each pair of primers, the 5'end of one of them is phosphorylated.
  • the above-mentioned primer pairs were used to amplify the specific fragment in Salmonella (product named SE-ps) and the specific fragment in E. coli O157 (product named O157-ps) (see primer table 6 for the sequence).
  • SE-ps specific fragment in Salmonella
  • E. coli O157-ps product named O157-ps
  • the PCR reaction program is 95°C for 2 minutes, and then 35 cycles of 98°C for 10s, 60°C for 15s, and 72°C for 10s. After the PCR is completed, the product is directly used in the Cas14 reaction.
  • reaction buffer of 10*Cas14 is: 250mM NaCl, 200mM HEPES, pH 7.5, 10mM DTT, 50% glycerol, and 50mM MgCl 2 .
  • reaction samples There are 2 reaction samples, the difference is that the added template is different, they are the PCR amplification products of SE-ps and O157-ps, and the reaction system with sterile water is used as a negative control.
  • the fluorescence quantitative PCR instrument used in this reaction is ABI StepOne Plus, and the detected signal is FAM fluorescence.
  • the results are shown in Figure 8. After subtracting the background signal (that is, adding sterile water as a template), the FAM fluorescence signal of the reaction group added with the SE-ps template is lower, while the FAM fluorescence signal of the reaction group added with the O157-ps template The signal grows very fast, which is significantly different from the signal strength of the SE-ps group. Based on the above results, it can be inferred that the SE-ps template cannot activate the cleavage of the O157-ps reporter nucleic acid probe, while the O157-ps template can activate the cleavage of the O157-ps reporter nucleic acid probe.
  • results of ROX fluorescence detection using a microplate reader show that the O157-ps template cannot activate the cleavage of the SE-ps reporter nucleic acid probe, while the SE-ps template can activate the cleavage of the SE-ps reporter nucleic acid probe.
  • the above results show that once the corresponding target exists in the detection system, the corresponding fluorescent signal can be detected, which is consistent with the expected result; on the contrary, the existence of a certain target can be judged according to the type of the detected fluorescent signal.

Abstract

L'invention concerne le domaine de détection d'acides nucléiques et un procédé de détection rapide multi-cible de procédé CRISPR, ainsi qu'un un kit de test associé. La présente invention concerne un système de détection utilisé pour détecter des molécules d'acides nucléiques cibles, contenant : (a) n sondes de combinaison d'ARN guide et d'acides nucléiques rapporteurs ayant une structure particulière ; et (b) une protéine Cas, la protéine Cas étant une protéine Cas ayant une activité de clivage collatérale d'acide nucléique simple brin.
PCT/CN2020/104588 2019-07-24 2020-07-24 Procédé de détection multi-cible crispr et kit de test associé WO2021013257A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US17/629,545 US20220267847A1 (en) 2019-07-24 2020-07-24 Crispr multi-target detection method and test kit therefor
CN202080052987.5A CN114174535A (zh) 2019-07-24 2020-07-24 Crispr多靶标检测方法及其试剂盒
EP20843278.1A EP4006170A4 (fr) 2019-07-24 2020-07-24 Procédé de détection multi-cible crispr et kit de test associé

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910673246.8A CN112301101A (zh) 2019-07-24 2019-07-24 Crispr多靶标检测方法及其试剂盒
CN201910673246.8 2019-07-24

Publications (1)

Publication Number Publication Date
WO2021013257A1 true WO2021013257A1 (fr) 2021-01-28

Family

ID=74193334

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/104588 WO2021013257A1 (fr) 2019-07-24 2020-07-24 Procédé de détection multi-cible crispr et kit de test associé

Country Status (4)

Country Link
US (1) US20220267847A1 (fr)
EP (1) EP4006170A4 (fr)
CN (2) CN112301101A (fr)
WO (1) WO2021013257A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113686934A (zh) * 2021-08-13 2021-11-23 广东海洋大学 一种CRISPR/Cas12a-RCA电化学传感器检测体系及其应用
CN114480384A (zh) * 2021-06-30 2022-05-13 山东舜丰生物科技有限公司 一种基于crispr技术检测口蹄疫病毒的方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113234801B (zh) * 2021-05-15 2023-05-02 浙江微景生物科技有限公司 一种CRISPR_Cas系统免标记核酸检测方法及试剂盒
CN115404268B (zh) * 2022-10-11 2023-04-07 芜湖森爱驰生物科技有限公司 一种sry基因检测探针和试剂盒
CN117126925A (zh) * 2023-08-07 2023-11-28 中科枢密生物技术(武汉)有限公司 一种能够长期稳定保存的Crispr-Cas核酸检测试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140212869A1 (en) * 2013-01-25 2014-07-31 Agilent Technologies, Inc. Nucleic Acid Proximity Assay Involving the Formation of a Three-way junction
US20150211058A1 (en) * 2014-01-29 2015-07-30 Agilent Technologies, Inc. CAS9-based Isothermal Method of Detection of Specific DNA Sequence
WO2019011022A1 (fr) * 2017-07-14 2019-01-17 上海吐露港生物科技有限公司 Application d'une protéine cas, procédé de détection d'une molécule d'acide nucléique cible et kit
CN109312336A (zh) * 2016-02-23 2019-02-05 阿克生物公司 用于靶标检测的方法和组合物

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5958034B2 (ja) * 2012-04-11 2016-07-27 富士レビオ株式会社 分子ビーコン型プローブを用いた標的核酸の検出方法
CN108220400A (zh) * 2018-01-02 2018-06-29 武汉启丰生物技术有限公司 一种基于CRISPR-Cas10核酸蛋白复合物的快捷核酸检测定量法
CN109837273B (zh) * 2018-09-17 2021-06-29 东南大学 一种crispr辅助dna靶向富集方法及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140212869A1 (en) * 2013-01-25 2014-07-31 Agilent Technologies, Inc. Nucleic Acid Proximity Assay Involving the Formation of a Three-way junction
US20150211058A1 (en) * 2014-01-29 2015-07-30 Agilent Technologies, Inc. CAS9-based Isothermal Method of Detection of Specific DNA Sequence
CN109312336A (zh) * 2016-02-23 2019-02-05 阿克生物公司 用于靶标检测的方法和组合物
WO2019011022A1 (fr) * 2017-07-14 2019-01-17 上海吐露港生物科技有限公司 Application d'une protéine cas, procédé de détection d'une molécule d'acide nucléique cible et kit

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALEXANDRA EAST-SELETSKY; MITCHELL R O’CONNELL; SPENCER C KNIGHT; DAVID BURSTEIN; JAMIE H D CATE; ROBERT TJIAN; JENNIFER A DOUDNA: "Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection", NATURE, vol. 538, 7624, 13 October 2016 (2016-10-13), pages 270 - 273, XP055719305, ISSN: 0028-0836, DOI: 10.1038/nature19802 *
FANG, RUI ET AL: "New Method of Genome Editing Derived From CRISPR/Cas9", PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS, vol. 40, no. 8, 31 August 2013 (2013-08-31), pages 691 - 702, XP055320441, ISSN: 0496-3490, DOI: 10.3724/SP.J.1206.2013.00215 *
JONATHAN S GOOTENBERG; OMAR O ABUDAYYEH; JEONG WOOK LEE; PATRICK ESSLETZBICHLER; AARON J DY; JULIA JOUNG; VANESSA VERDINE; NINA DO: "Nucleic acid detection with CRISPR-Cas13a/C2c2", SCIENCE, vol. 356, no. 2336, 28 April 2017 (2017-04-28), pages 438 - 442, XP055481345, ISSN: 0036-8075, DOI: 10.1126/science.aam9321 *
LI JUN, ZHANG YI, CHEN KUN-LING, SHAN QI-WEI, WANG YAN-PENG, LIANG ZHEN, GAO CAI-XIA: "CRISPR/Cas: a novel way of RNA-guided genome editing", HEREDITAS, vol. 35, no. 11, 14 October 2013 (2013-10-14), pages 1265 - 1273, XP055775588, ISSN: 0253-9772, DOI: 10.3724/SP.J.1005.2013.01265 *
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480384A (zh) * 2021-06-30 2022-05-13 山东舜丰生物科技有限公司 一种基于crispr技术检测口蹄疫病毒的方法
CN114480384B (zh) * 2021-06-30 2024-03-08 山东舜丰生物科技有限公司 一种基于crispr技术检测口蹄疫病毒的方法
CN113686934A (zh) * 2021-08-13 2021-11-23 广东海洋大学 一种CRISPR/Cas12a-RCA电化学传感器检测体系及其应用

Also Published As

Publication number Publication date
US20220267847A1 (en) 2022-08-25
EP4006170A4 (fr) 2023-08-30
EP4006170A1 (fr) 2022-06-01
CN112301101A (zh) 2021-02-02
CN114174535A (zh) 2022-03-11

Similar Documents

Publication Publication Date Title
WO2021013257A1 (fr) Procédé de détection multi-cible crispr et kit de test associé
CN111094588B (zh) 一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒
CN110551800B (zh) 一种耐高温Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒
US9487824B2 (en) Methods and compositions for enrichment of nucleic acids in mixtures of highly homologous sequences
EP2046989B1 (fr) Oligonucléotides spécialisés et leur utilisation pour l'amplification et la détection d'acides nucléiques
JP2009506788A (ja) 核酸の等温増幅のための方法、組成物及びキット
KR20110106922A (ko) 단일 세포 핵산 분석
US10704087B2 (en) Cooperative primers, probes, and applications thereof
CN106574304B (zh) 基于链侵入的dna扩增方法
JP2008529528A (ja) ポリヌクレオチドの検出用のキープローブ組成物および方法
KR102324117B1 (ko) 가닥 침입에 기초한 증폭에 의한 핵산 검출
US20200172958A1 (en) Multiplex probes
JP2015522292A (ja) 協働的プライマー、プローブ、およびそれらの用途
KR20090067334A (ko) 핵산 중합효소의 뉴클레아제 활성을 이용한 타깃핵산분자의 검출방법
US11584955B2 (en) Application of Cas protein, method for detecting target nucleic acid molecule and kit
CN105705660B (zh) 使用重叠引物和熔解探针检测单核苷酸多态性
WO2022121754A1 (fr) Procédé de détection de l'activité d'une ou de plusieurs polymérases
WO2022120914A1 (fr) Procédé de mesure de la longueur d'un produit d'amplification d'une ou de plusieurs molécules d'acide nucléique dans un échantillon
EP3856931B1 (fr) Conception spécifique d'allèles d'amorces coopératives pour génotypage de variants d'acide nucléique amélioré
WO2012046140A1 (fr) Mélange d'enzymes
JP2017521083A (ja) リステリア・モノサイトゲネス（Ｌｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓ）を検出するための配列およびこの配列の使用
KR20230124697A (ko) SARS-CoV-2 변이를 검출하는 방법
US20160326582A1 (en) Method and kit for target molecule characterization
WO2013140339A1 (fr) Contrôle positif pour pcr

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20843278

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020843278

Country of ref document: EP

Effective date: 20220224